Adjacent Non-tumor Lung Tissues Research Articles

Overexpression and proliferation dependence of acyl-CoA thioesterase 11 and 13 in lung adenocarcinoma.

The metabolites of fatty acyl-Coenzyme A (CoA) and metabolic enzymes contribute to lipid biosynthesis, signal transduction, and gene transcription. Previous studies have indicated that elevated concentrations of specific free fatty acids in the plasma and overexpression of specific fatty acyl-CoA metabolic enzymes are observed in patients with lung adenocarcinoma. However, there are >30 enzymes in this metabolic network and have been fully investigated. In the present study, the expression levels of enzymes in the acyl-CoA synthetase (ACS) and acyl-CoA thioesterase (ACOT) families were analyzed from six microarray expression datasets that were collected from Gene Expression Omnibus. Compared with adjacent non-tumor lung tissue, lung adenocarcinoma tissue exhibited significantly higher ACOT11 and ACOT13 expression. Kaplan-Meier plotter database analysis demonstrated that high levels of ACOT11 and ACOT13 were associated with a worse overall survival rate. The proliferation of the lung adenocarcinoma cell lines CL1-0 and CL1-5 was inhibited when ACOT11 and ACOT13 were downregulated by short hairpin RNA. Although ACOT11 and ACOT13 knockdown did not significantly affect the total amount of intracellular and medium-free fatty acids, ACOT11 and ACOT13 knockdown-mediated growth inhibition was rescued by the addition of fatty acids. In conclusion, ACOT11 and ACOT13 were upregulated in clinical specimens of lung adenocarcinoma, which may contribute to increased cell proliferation through the increased availability of fatty acids. The metabolites of the two enzymes may be critical for development of lung adenocarcinoma.

HBXIP suppression reduces cell proliferation and migration and its overexpression predicts poor prognosis in non-small-cell lung cancer.

Emerging evidence has demonstrated that the high expression of HBXIP has been correlated with many cancers. With evaluation of the functional role of HBXIP in non-small-cell lung cancer, the primary aim of this study is to investigate the correlation between HBXIP expression and the prognosis of non-small-cell lung cancer patients. The protein levels of HBXIP were detected using western blotting in non-small-cell lung cancer cells. Cell proliferation and migration assays were measured to evaluate the function of HBXIP in non-small-cell lung cancer cells. A total of 120 non-small-cell lung cancer patients with strict follow-up and 60 adjacent non-tumor lung tissues were selected for immunohistochemical staining of the HBXIP protein. The localization of the HBXIP protein was detected in A549 non-small-cell lung cancer cells using immunofluorescence staining. The correlation between HBXIP expression and the clinicopathological features of non-small-cell lung cancer patients was analyzed by a chi-squared and Fisher's exact test. The overall survival rates of all of the non-small-cell lung cancer patients were calculated using the Kaplan-Meier method, and univariate and multivariate analyses were performed using the Cox proportional hazards regression model. In function, we showed that suppression of HBXIP decreased A549 cell proliferation and migration. HBXIP protein showed a mainly cytoplasmic staining pattern in non-small-cell lung cancer using immunohistochemical staining in paraffin-embedded non-small-cell lung cancer tissues and immunofluorescence staining in A549 cells. The HBXIP protein had strong positive staining in the non-small-cell lung cancer tissues, which was significantly higher than the percentage of adjacent non-tumor tissues. The overexpression of HBXIP was closely correlated with histological grade, clinical stage, lymph node metastasis, and lower overall survival rates of patients with non-small-cell lung cancer. Moreover, multivariate analysis suggested that HBXIP emerged as a significant independent prognostic factor along with clinical stage in patients with non-small-cell lung cancer. In conclusion, a high level of expression of HBXIP is associated with the progression of non-small-cell lung cancer and may be a useful biomarker for poor prognostic evaluation and a potential molecular therapy target for patients with non-small-cell lung cancer.

Combination of CALR and PDIA3 is a potential prognostic biomarker for non-small cell lung cancer.

Proteomic-based approaches for biomarker discovery are promising strategies used in cancer research. In this study, we performed quantitative proteomic analysis on 16 paired samples of non-small cell lung cancer (NSCLC) and adjacent non-tumor lung tissues using label-free quantitative proteomics and liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS) to identify differentially expressed proteins. A total of 91 proteins were differentially expressed in NSCLC compared with adjacent non-tumor lung tissues among 4047 identified proteins (fold change > 1.5 or < 0.67, P < 0.05). Gene ontology (GO) analysis, Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis and ingenuity pathway analysis (IPA) of 91 dysregulated proteins showed that they were related to the cancer-associated biological processes. We confirmed that the candidate proteins, calreticulin (CALR) and protein disulfide isomerase family A member 3 (PDIA3) were overexpressed in NSCLC by real-time PCR using 20 paired samples and western blot using 5 paired samples. PDIA3 expression was highly associated with CALR expression (Spearman r = 0.345, P = 0.001) and they were co-localized and interacted with each other in A549 and H460 cells. Moreover, survival analysis performed in tissue microarray with 88 samples indicated that low expression of both CALR and PDIA3 in NSCLC was positively associated with poor overall survival. Combination of CALR and PDIA3 might serve as an efficient biomarker and improved the prediction of NSCLC prognosis significantly (P = 0.023). Our results collectively provide a potential biomarker dataset for NSCLC prognosis, especially the prognostic value of combined expression of CALR and PDIA3.

DNA methylation profiling identifies the HOXA11 gene as an early diagnostic and prognostic molecular marker in human lung adenocarcinoma.

DNA hypermethylation plays important roles in carcinogenesis by silencing key genes. The goal of our study was to identify pivotal genes using MethyLight and assessed their diagnostic and prognostic values in lung adenocarcinoma (AD). In the present study, we detected DNA methylation at sixteen loci promoter regions in twenty one pairs of primary human lung AD tissues and adjacent non-tumor lung (AdjNL) tissues using the real-time PCR (RT-PCR)-based method MethyLight. By comparing the sixteen analyzed loci in lung AD tissues and AdjNL and non-tumor (NL) tissues, we found that, among the six genes identified with hypermethylation, the HOXA11, CDKN2A-EX2 and EYA4 genes showed highly promising DNA hypermethylation diagnostic markers in the lung AD tissues. Moreover, comparing lung AD tissues (> 2 cm in diameter) to the AdjNL or AD in situ (AIS) tissues by RT-qPCR and immunohistochemistry revealed that HOXA11 expression was significantly increased. A further study showed that HOXA11 expression was controlled by methylation in the promoter region in human lung tumor cell lines. Aberrant hypermethylation and the methylation-induced down-regulation of HOXA11 may promote tumor progression. Our results suggested that HOXA11 might be a diagnostic and prognostic marker in patients with lung AD.

Transcriptomic Microenvironment of Lung Adenocarcinoma.

Background: Tissues surrounding tumors are increasingly studied to understand the biology of cancer development and identify biomarkers.Methods: A unique geographic tissue sampling collection was obtained from patients that underwent curative lobectomy for stage I pulmonary adenocarcinoma. Tumor and nontumor lung samples located at 0, 2, 4, and 6 cm away from the tumor were collected. Whole-genome gene expression profiling was performed on all samples (n = 5 specimens × 12 patients = 60). Analyses were carried out to identify genes differentially expressed in the tumor compared with adjacent nontumor lung tissues at different distances from the tumor as well as to identify stable and transient genes in nontumor tissues with respect to tumor proximity.Results: The magnitude of gene expression changes between tumor and nontumor sites was similar with increasing distance from the tumor. A total of 482 up- and 843 downregulated genes were found in tumors, including 312 and 566 that were consistently differentially expressed across nontumor sites. Twenty-nine genes induced and 34 knocked-down in tumors were also identified. Tumor proximity analyses revealed 15,700 stable genes in nontumor lung tissues. Gene expression changes across nontumor sites were subtle and not statistically significant.Conclusions: This study describes the transcriptomic microenvironment of lung adenocarcinoma and adjacent nontumor lung tissues collected at standardized distances relative to the tumor.Impact: This study provides further insights about the molecular transitions that occur from normal tissue to lung adenocarcinoma and is an important step to develop biomarkers in nonmalignant lung tissues. Cancer Epidemiol Biomarkers Prev; 26(3); 389-96. ©2016 AACR.

Expression of RNA-binding motif 10 is associated with advanced tumor stage and malignant behaviors of lung adenocarcinoma cancer cells.

This study assessed RNA-binding motif 10 expression in lung adenocarcinoma tissues and examined the role and mechanism of RNA-binding motif 10 in the regulation of lung adenocarcinoma malignancy. Lung adenocarcinoma and corresponding adjacent non-tumor lung tissues from 41 patients were subjected to reverse transcription-polymerase chain reaction and Western blot assessment to detect RNA-binding motif 10 expression. Recombinant lentivirus carrying RNA-binding motif 10 complementary DNA was used to infect lung adenocarcinoma cell lines, A549 and H1299 cells. Complementary DNA microarray was used to profile RNA-binding motif 10-regulated genes. Levels of RNA-binding motif 10 messenger RNA and protein were significantly lower in lung adenocarcinoma tissues than those in paired non-tumor tissues (p < 0.001). Reduced RNA-binding motif 10 expression was found to be associated with an advanced tumor stage. RNA-binding motif 10 overexpression inhibited viability and colony formation capacity of lung adenocarcinoma cell lines and induced cell-cycle arrest at G0/G1 phase in A549 cells and at S phase in H1299 cells. Complementary DNA microarray analysis identified 304 upregulated and 386 downregulated genes induced by RNA-binding motif 10 overexpression, which may be involved in cancer, focal adhesion, peroxisome proliferator-activated receptor-regulated gene pathway, cytokine-cytokine receptor interaction, mitogen-activated protein kinase signaling, complement and coagulation cascades, platelet amyloid precursor protein pathway, extracellular matrix-receptor interaction, and small cell lung cancer-related genes. Expression of FGF2, EGFR, WNT5A, NF-κB, and RAP1A was downregulated, whereas expression of AKT2, BIRC3, and JUN was upregulated. RNA-binding motif 10 messenger RNA and protein were reduced in lung adenocarcinoma tissues, and RNA-binding motif 10 overexpression inhibited lung adenocarcinoma cancer cell malignant behavior in vitro. Molecularly, RNA-binding motif 10 regulates many gene pathways involving in the tumor development or progression.

MicroRNA-300 targets hypoxia inducible factor-3 alpha to inhibit tumorigenesis of human non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most deadly human cancers. MicroRNA-300 acts as both tumor promoter and suppressor in different types of cancer. Here, we try to identify the function of microRNA-300 in human NSCLC. We compared MicroRNA-300 levels between tumor tissues versus paired adjacent non-tumor lung tissues from NSCLC patients, and in NSCLC versus normal lung cell lines. Effects of microRNA-300 on cell proliferation, invasion and migration were examined in vitro, and on tumor growth in vivo using a xenograft mouse model. Potential mRNA targets of microRNA-300 were predicted and underlying mechanism was explored. MicroRNA-300 expression was lower in both NSCLC tissues and cell lines. Overexpression of microRNA-300 inhibited proliferation, invasion and migration of NSCLC cells in vitro, and tumor growth in vivo. MicroRNA-300 could directly bind to the 3'-UTR of hypoxia inducible factor-3 alpha (HIF3α) mRNA, and inhibit both its mRNA and protein expressions. Restoring HIF3α expression could rescue the inhibitory effects of microRNA-300 on tumorigenesis of NSCLC both in vitro and in vivo. MicroRNA-300 is a tumor suppressor microRNA in NSCLC by downregulating HIF3α expression. Both microRNA-300 and HIF3α may serve as potential therapeutic targets in NSCLC treatment.

MiR-99a regulates ROS-mediated invasion and migration of lung adenocarcinoma cells by targeting NOX4.

miR-99a is frequently downregulated in various types of human malignancies including lung adenocarcinoma. Recent studies have reported that miR-99a regulates cell growth and cell cycle progression by targeting mTOR, AKT1 and FGFR3. However, the underlying mechanisms involved in the modulation of invasion and migration by miR-99a remain elusive. In this study, we analyzed the relationship between the expression of miR-99a and clinical stage or metastasis in 90 matched lung adenocarcinoma and adjacent non-tumor lung tissues. Downregulation of miR-99a was significantly associated with advanced stage and tumor metastasis in lung adenocarcinoma patients, and it was found to be a poor prognostic factor in lung adenocarcinoma. Furthermore, functional experiments found that overexpression of miR-99a inhibited the proliferation, migration and invasion of lung adenocarcinoma A549 and Calu3 cells in vitro. We then identified NOX4 as a target gene of miR-99a and NOX4 mediated the inhibition of invasion and migration of lung adenocarcinoma cells by miR-99a. By targeting NOX4-mediated ROS production, miR-99a regulated the invasion and migration of lung adenocarcinoma cells. Moreover, overexpression of miR-99a significantly inhibited tumor growth in vivo. Immunohistochemical staining analysis of the mouse tumor tissues revealed that NOX4 levels were downregulated in the miR-99a treatment group, confirming the in vitro data of NOX4 as a direct target gene of miR-99a. Taken together, these data indicate for the first time that miR-99a directly regulates the invasion and migration in lung adenocarcinoma by targeting NOX4 and that overexpression of miR-99a may become a therapeutic strategy for lung adenocarcinoma.

MiR-198 targets SHMT1 to inhibit cell proliferation and enhance cell apoptosis in lung adenocarcinoma.

MiR-198 is involved in tumorigenesis, migration, invasion, and metastasis of various malignant cancers. However, the exact expression levels of miR-198 and the molecular mechanism underlying its role in lung adenocarcinoma require further exploration. In this study, quantitative real-time PCR was applied to study miR-198 and serine hydroxymethyltransferase 1 (SHMT1) expression in 47 paired lung adenocarcinoma tissues and adjacent nontumor lung tissues. Clinicopathological characters were analyzed. Pearson's correlation analysis was used to detect the relationship between miR-198 and SHMT1 expression. The function of miR-198 was explored by measuring cell proliferation, cell apoptosis, and the cell-cycle in vitro and in vivo. The target gene of miR-198 was certified using dual luciferase report assay. We found that in lung adenocarcinoma, miR-198 was significantly downregulated and SHMT1 was inversely upregulated. A strong negative correlation was noticed between miR-198 and SHMT1 expression. Further analysis revealed that miR-198 expression was associated with TNM stage and lymph node metastasis. Upregulated miR-198 could inhibit cell proliferation, enhance cell apoptosis, and lead to cell-cycle arrest in lung adenocarcinoma, which showed a more effective alteration than SHMT1 siRNA. Moreover, we identified SHMT1 as a target gene of miR-198. In conclusion, miR-198 suppressed proliferation of lung adenocarcinoma cells both in vitro and in vivo by directly targeting SHMT1. miR-198 may be a potential therapeutic target for lung adenocarcinoma in the near future.

Significance of DEK overexpression for the prognostic evaluation of non-small cell lung carcinoma.

In the present study, we explored the role of DEK expression for the prognostic evaluation of non-small cell lung carcinoma (NSCLC). DEK protein and mRNA expression levels were detected in NSCLC cells and fresh tissue samples of NSCLC paired with adjacent non-tumor tissues, respectively. NSCLC cases (n=196) meeting strict follow-up criteria were selected for immunohistochemical staining of DEK protein. Correlations between DEK expression and clinicopathological features of the NSCLC cases were evaluated using Chi-square tests. Survival rates were calculated using the Kaplan-Meier method, and the relationship between prognostic factors and patient overall survival was analyzed using Cox proportional hazard analysis. Based on the results, the levels of DEK protein and mRNA were significantly upregulated in 6 fresh tissue samples of NSCLC. Immunohistochemical analysis showed that the DEK expression rate was significantly higher in the NSCLC samples compared with either the adjacent non-tumor tissues or normal lung tissues. DEK expression was correlated with poor differentiation and late pathological stage of NSCLC. DEK expression was also correlated with low disease-free survival and overall survival rates. In the early-stage group, disease-free and overall survival rates of patients with DEK expression were significantly lower than those of patients without DEK expression. Further analysis using a Cox proportional hazard regression model revealed that DEK expression emerged as a significant independent hazard factor for the overall survival rate of patients with NSCLC. Consequently, DEK plays an important role in the progression of NSCLC. DEK may potentially be used as an independent biomarker for the prognostic evaluation of NSCLC.

Silencing of miR-137 by aberrant promoter hypermethylation in surgically resected lung cancer.

Recent studies demonstrated that miR-137 is downregulated in various tumors, and that it functions as a tumor suppressor. miR-137 could be silenced by its aberrant promoter hypermethylation. The purpose of this study was to investigate the significance of MIR137 promoter methylation on its expression in lung cancer. Lung cancer cell lines were treated with either a DNA methyltransferase inhibitor (5-azacytidine, AZA) and/or an HDAC inhibitor (trichostatin A, TSA) to determine whether miR-137 expression was reactivated. Paired lung tumor and adjacent non-tumor lung tissues were obtained (n=50). Quantitative methylation-specific PCR and bisulfite sequencing were used to analyze the methylation status of MIR137, and real-time RT-PCR was performed to analyze miR-137 expression. miR-137 was reactivated by treatment with either AZA and/or TSA in lung cancer cell lines. Methylation-specific PCR showed increased MIR137 promoter methylation in lung tumors compared with adjacent non-tumor tissues, which was further validated by bisulfite sequencing. The expression of miR-137 was downregulated significantly in lung tumors, which was correlated with level of MIR137 promoter methylation inversely. miR-137 downregulation was related to its promoter hypermethylation in lung cancer. Further studies are needed to assess its value as a prognostic factor and potential therapeutic applications in lung cancer.

Clinical implications of insulin-like growth factor II mRNA-binding protein 3 expression in non-small cell lung carcinoma.

In order to examine the role of insulin-like growth factor II mRNA-binding protein 3 (IMP3) expression for the prognostic evaluation of non-small cell lung carcinoma (NSCLC), a total of 186 breast cancer patients, with adjacent non-tumor lung tissues, were selected for immunohistochemical staining of IMP3 protein. The NSCLC tissues and paired adjacent non-tumor tissues of six patients were quantified using reverse transcription quantitative polymerase chain reaction. The correlations between IMP3 overexpression and the clinical features of NSCLC were evaluated using the χ2 test and Fisher’s exact test. The survival rate was calculated using the Kaplan-Meier method, and the association between prognostic factors and patient survival was also analyzed by Cox’s proportional hazards models. The results showed that IMP3 protein exhibited a mainly cytoplasmic staining pattern in the NSCLC tissues. The positive rate of IMP3 protein expression was 74.7% (139/186) in the NSCLC tissues and was significantly higher than the rate of 19.9% (37/186) in the adjacent non-tumor tissues. The expression rate of the NQO1 protein was correlated with a large tumor size, poor differentiation, lymph node metastasis, late clinical stage, and disease-free and overall survival rates in the NSCLC patients. In the early- and late-stage NSCLC groups, the disease-free and overall survival rates of the patients with IMP3 expression were significantly lower than those of the patients without IMP3 expression. Further analysis using Cox’s proportional hazard regression model revealed that IMP3 expression was a significant independent hazard factor for the overall survival rate of patients with NSCLC. In conclusion, the present study found that IMP3 plays a significant role in the progression of NSCLC, and that it may potentially be used as an independent biomarker for prognostic evaluation of the cancer.

MicroRNA-15a induces cell apoptosis and inhibits metastasis by targeting BCL2L2 in non-small cell lung cancer.

MicroRNAs (miRNAs) play a critical role in cancer development and progression. Aberrant expression of miR-15a has recently been reported in several cancers, but its role in non-small cell lung cancer (NSCLC) still remains obscure. We investigated the effects of miR-15a on proliferation, apoptosis, and metastasis in A549 cells. Eighteen paired NSCLC and adjacent non-tumor lung tissues were surgically removed and immediately snap frozen until total RNA was extracted and confirmed by two independent pathologists. The targets of miR-15a were predicted by bioinformatics tools. RNA isolation and quantitative real-time PCR (qRT-PCR), Western blot analysis, cell proliferation assay, cell cycle analysis, cell apoptosis assay, and migration and invasion assays were done. The wild type (WT) or mutant type (MT) 3'-untranslated region (UTR) vectors were co-transfected with miR-15a or negative control into A549 cells, and after 24 h of transfection, luciferase activity was measured using the Dual-Glo luciferase assay kit. Statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL, USA). P values of less than 0.05 were considered statistically significant. miR-15a was significantly downregulated in NSCLC than in adjacent non-cancerous tissues. miR-15a overexpression remarkably inhibited cell viability, invasion, and migration and promoted the apoptosis of NSCLC cells. Additionally, inhibition of miR-15a expression had the opposite effects on tumor progression, while cell cycle remained unaltered. Furthermore, we identified that BCL2L2 was a target of miR-15a and negatively regulated by miR-15a at the translational level. miR-15a acts as a tumor suppressor in NSCLC by directly targeting BCL2L2 and may serve as a potential diagnostic biomarker and therapeutic target for NSCLC.

Astrocyte Elevated Gene-1 Increases Invasiveness of NSCLC Through Up-Regulating MMP7

Background/Aims: The aggressive manner of non-small cell lung cancer (NSCLC) cells accounts for the majority of the lethality of the disease. Recently, increased astrocyte elevated gene-1 (AEG-1) levels have been shown to closely correlate with poor prognosis of NSCLC, whereas the underlying mechanisms are not clear. Methods: We examined the AEG-1 and matrix metalloproteinase 7 (MMP7) levels in NSCLC tissues, compared to the paired adjacent non-tumor lung tissue. We modulated AEG-1 levels in NSCLC cells, and examined its effects on MMP7 levels by RT-qPCR, on cellular protein by Western blot, and on secreted protein by ELISA. We also examined the cell invasiveness in AEG-1-modified NSCLC cells in a transwell cell migration assay. We used specific signal pathway inhibitors to treat AEG-1-modified NSCLC cells and examined its effects on MMP7. Results: AEG-1 and MMP7 levels were both significantly increased in NSCLC tissues, compared to the paired adjacent non-tumor lung tissue. The AEG-1 and MMP7 levels were strongly correlated. Overexpression of AEG-1 in NSCLC cells significantly increased MMP7 levels and cell invasiveness, while AEG-1 depletion in NSCLC cells significantly decreased MMP7 levels and cell invasiveness. Application of a specific MAPK-p42/p44 inhibitor, but not application of specific inhibitors for MAPK-p38, PI3k/Akt, or JNK signaling pathways, to AEG-1-overexpressing NSCLC cells substantially abolished the AEG-1-mediated MMP7 up regulation. Conclusion: AEG-1 promotes NSCLC cell invasiveness through MAPK-p42/p44-dependent activation of MMP7.

Gene expression profiles on predicting protein interaction network and exploring of new treatments for lung cancer.

In the present study, we aimed to explore disease-associated genes and their functions in lung cancer. We downloaded the gene expression profile GSE4115 from Gene Expression Omnibus (GEO) database. Total 97 lung cancer and 90 adjacent non-tumor lung tissue (normal) samples were applied to identify the differentially expressed genes (DEGs) by paired t test and variance analysis in spectral angle mapper (SAM) package in R. Gene Ontology (GO) functional enrichment analysis of DEGs were performed with Database for Annotation Visualization and Integrated Discovery, followed by construction of protein-protein interaction (PPI) network from Human Protein Reference Database (HPRD). Finally, network modules were analyzed by the MCODE algorithm to detect protein complexes in the PPI network. Total 3,102 genes were identified as DEGs at FDR < 0.05, including 1,146 down-regulated and 1,956 up-regulated DEGs. GO functional enrichment analysis revealed that up-regulated DEGs mainly participated in cell cycle and intracellular related functions, and down-regulated DEGs might influence cell functions. There were 39,240 pairs of PPIs in human obtained from HPRD databases, 3,102 DEGs were mapped to this PPI network, in which 2,429 pairs of PPIs and 1,342 genes were identified. With MCODE algorithm, 48 modules were selected, including five corresponding modules and three modules with differences in gene expressing profiles. In addition, three DGEs, FXR2, ARFGAP1 and ELAVL1 were discovered as potential lung cancer related genes. The discovery of featured genes which were probably related to lung cancer, has a great significance on studying mechanism, distinguishing normal and cancer tissues, and exploring new treatments for lung cancer.

Gene expression analysis of lung adenocarcinoma and matched adjacent non-tumor lung tissue.

The aim of this study was to find disease-associated genes and gene functions in lung adenocarcinoma and matched adjacent non-tumor lung tissues with DNA microarray. We downloaded the gene expression profile GSE32863 from the Gene Expression Omnibus database including 58 lung adenocarcinoma and 58 adjacent non-tumor lung tissue samples. Data were preprocessed and the differentially expressed genes (DEGs) were identified using packages in the R computing language. The selected DEGs were further analyzed with bioinformatics methods. After the coexpression network of DEGs was constructed by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins), we analyzed gene functions with DAVID (The Database for Annotation, Visualization and Integrated Discovery) and WebGestalt (WEB-based Gene Set Analysis Toolkit). A total of 1429 genes were filtered as DEGs, including 873 downregulated genes and 556 upregulated genes, and the DEGs including CDC45, CCNB2, CDC20, MCM2, PTTG1, MCM4 and FEN1 were most significantly related to cell cycle and DNA replication. The discovery of featured genes which were significantly related to cell cycle and DNA replication has potential for use in the clinic for the diagnosis of lung adenocarcinoma in the future. However, further experiments will be needed to confirm our result.

Abstract B24: A systematic search for cancer/testis antigens in lung cancer identifies VCX/Y genes expanding the repertoire of potential immunotherapeutic targets.

Abstract Background: A wide range of therapeutic cancer vaccines that target diverse tumor-associated antigens are currently been evaluated in early phase clinical trials. However the effectiveness of cancer vaccines is so far limited, potentially due in part to the limited availability of candidate targets with broad expression in a tumor type. Cancer/testis (CT) antigens are a major target of cancer vaccines, because CT antigens are specifically expressed in immune-privileged testis and placenta, and exhibit aberrant expression in various types of cancer, resulting in high immunogenicity with no self-tolerance and reduced adverse effects. However expression of particular antigens is limited to a subset of tumors of a given type. Thus there is a need to identify antigens with complementary expression patterns for more effective therapeutic intervention. Methods: Gene expression data of BioGPS, and DNA methylation and RNA-seq data of TCGA lung cancer samples were downloaded. All lung cancer cell lines were cultured in RPMI1640 containing 10% FBS. A total of 83 pairs of treatment naive lung adenocarcinoma tissues and adjacent non-tumor lung tissues had been obtained earlier with informed consent. Gene expression data for cell lines and tissues were obtained using the Illumina Human WG-6 v3.0 Expression BeadChips. For mass spectrometric analysis, 38 non-small cell lung cancer (NSCLC) cell lines were cultured according to the standard SILAC protocol. The same batch of cells was used for analysis of whole cell extract, conditioned media and cell surface proteins. Tissue microarrays comprised 463 surgically resected NSCLC tumor specimens collected under an IRB protocol. Monoclonal anti-mouse VCX3A antibody was used for Western blot and immunohistochemistry analysis. Results: With mining the public database BioGPS, we identified VCX/Y gene family as novel CT antigens in lung cancer. Our findings also suggested epigenetic regulation of VCX/Y gene expression, and potential oncogenic roles of VCX/Y genes. Expression of one of the VCX/Y family genes, VCX3A, was further confirmed at the protein level in ~20% of lung adenocarcinoma and ~35% of squamous cell carcinoma tumors. We further examined gene expression patterns of CT antigens to determine whether a panel could be assembled with broad representation in lung adenocarcinoma. Of 255 known CT antigens, 109 CT antigens were profiled at both mRNA and protein levels in 38 NSCLC cell lines. mRNA expression levels of 10 CT antigens were significantly and positively correlated with protein expression levels. We examined mRNA expression profiles of these 10 CT antigens, together with VCX and MAGEA3 which is a current target for vaccine immunotherapy, in 83 lung adenocarcinoma tumors. We selected XAGE1, VCX, IL13RA2, and SYCE1 to form a CT antigen panel and compared mRNA expression levels of these four genes between tumor and adjacent non-tumor tissue. One or more of the CT antigens in the panel were overexpressed in 66 (79.5%) of lung adenocarcinomas. Conclusions: The present study identifies for the first time a novel CT antigen VCX/Y gene family in lung cancer. A CT antigen panel was found to provide a broad coverage of lung adenocarcinoma tumors tested, suggestive of a potential for more effective immunotherapy through targeting the combined antigen panel compared to a single antigen target. Citation Format: Ayumu Taguchi, Allen D. Taylor, Jaime Rodriguez, Muge Celiktas, Hui Liu, Xiaotu Ma, Qing Zhang, Chee-Hong Wong, Alice Chin, Luc Girard, Carmen Behrens, Wan Lam, Stephen Lam, John D. Minna, Ignacio I. Wistuba, Adi F. Gazdar, Samir M. Hanash. A systematic search for cancer/testis antigens in lung cancer identifies VCX/Y genes expanding the repertoire of potential immunotherapeutic targets. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B24.

Gene silencing of SLC5A8 identified by genome-wide methylation profiling in lung cancer

BackgroundAberrant DNA hypermethylation has been implicated as a component of an epigenetic mechanism that silences genes in cancers. MethodsWe performed a genome-wide search to identify differentially methylated loci between 26 tumor and adjacent non-tumor paired tissues from same lung cancer patients using restriction landmark genomic scanning (RLGS) analysis. Among 229 loci which were hypermethylated in lung tumors as compared to adjacent non-tumor tissues, solute carrier family 5, member 8 (SLC5A8) was one of the hypermethylated genes, and known as a tumor suppressor gene which is silenced by epigenetic changes in various tumors. We investigated the significance of DNA methylation in SLC5A8 expression in lung cancer cell lines, and 23 paired tumor and adjacent non-tumor lung tissues by reverse transcription-PCR (RT-PCR), quantitative methylation specific PCR (QMSP) and bisulfite modified DNA sequencing analyses. ResultsReduced or lost expression of SLC5A8 was observed in 39.1% (9/23) of the tumor tissues as compared with paired adjacent non-tumor tissues. Bisulfite sequencing results of lung cancer cell lines and tissues which did not express SLC5A8 showed a densely methylated promoter region of SLC5A8. SLC5A8 was reactivated by treatment with DNA methyltransferase inhibitor, 5-Aza and/or HDAC inhibitor, trichostatin A (TSA) in lung cancer cell lines, which did not express SLC5A8. Hypermethylation was detected at the promoter region of SLC5A8 in primary lung tumor tissues as compared with adjacent non-tumor tissues (14/23, 60.9%). ConclusionThese results suggest that DNA methylation in the SLC5A8 promoter region may suppress the expression of SLC5A8 in lung tumor.

Expression and bioinformatic analysis of ornithine aminotransferase in non-small cell lung cancer

背景与目的已有的研究表明，鸟氨酸氨基转移酶（ornithine aminotransferase, OAT）可能参与多种恶性肿瘤的发生和发展，本研究旨在检测非小细胞肺癌（non-small cell lung cancer, NSCLC）中OAT mRNA和蛋白质的表达，并对其进行生物信息学分析。方法通过RT-PCR的方法比较A549和16HBE细胞间OAT mRNA水平的差异；采用免疫组织化学SP法检测55例肺癌组织和17例癌旁肺组织中OAT蛋白表达；利用生物信息学的方法对OAT蛋白的生物信息学特征和相互作用蛋白进行分析，并对筛选出来的相互作用蛋白进行GO注释和信号通路分析。结果① A549中OAT mRNA相对含量较16HBE中低，两者差异约2.85倍。②NSCLC中OAT蛋白的表达明显高于癌旁肺组织（P < 0.05）；OAT蛋白在鳞癌和腺癌间的表达差异有统计学意义，而与患者性别、年龄、有无淋巴结转移、肿瘤直径及TNM分期无关。③生物信息学分析提示OAT蛋白定位于线粒体、为高度保守的亲水性蛋白，具有Aminotran-3结构域，可能存在多个丝/苏氨酸磷酸化位点，在信号转导、转录和分子转运等方面发挥一定的作用；在筛选出来的54种可能和OAT存在相互作用的蛋白质中，TNF和TRAF6这两种蛋白参与了NF-κB信号通路。结论OAT可能在NSCLC的发生和发展中发挥重要作用，有望成为肺癌的早期诊断标志物和治疗的新靶点。

MicroRNA-101 Exerts Tumor-Suppressive Functions in Non-small Cell Lung Cancer through Directly Targeting Enhancer of Zeste Homolog 2

Overexpression of the enhancer of zeste homolog 2 (EZH2) protein has been found in broad range of cancer types, including non-small cell lung cancer (NSCLC). Nevertheless, the mechanisms by which EZH2 becomes overexpressed in NSCLC remain unclear. MicroRNAs (miRNAs) can regulate target gene expression through translational control. In this study, we investigate whether miRNA (miR-101) regulates EZH2 expression in NSCLC. We evaluated the expression of miR-101 and EZH2 in 20 matched NSCLC and adjacent nontumor lung tissues by reverse-transcriptase polymerase chain reaction and immunohistochemistry, respectively. Luciferase reporter assay was used to determine whether miR-101 directly targets EZH2. To assess the effect of miR-101 on NSCLC biological behavior, cell proliferation, invasion, and response to chemotherapy were analyzed using NSCLC cells transfected with miR-101 mimics or transfected with specific small interfering RNA to deplete EZH2 (small interfering RNA-EZH2). Reduced expression of miR-101 was associated with overexpression of EZH2 in NSCLC tumor tissues. Transfection of miR-101 mimics significantly suppressed the activity of the luciferase reporter containing wild type but not mutant EZH2 3'-UTR and decreased EZH2 expression in NSCLC cell lines. Furthermore, enforced expression of miR-101 or knockdown of EZH2 led to reduced NSCLC cell proliferation and invasion and sensitized cancer cells to paclitaxel-mediated apoptosis through inducing expression of the proapoptotic protein Bim. miR-101 inhibits cell proliferation and invasion and enhances paclitaxel-induced apoptosis in NSCLC cells, at least in part, by directly repressing EZH2 expression. Therapeutic strategies to rescue miR-101 expression or silence EZH2 may be beneficial to patients with NSCLC in the future.